No abstract

Myeloperoxidase (MPO), a member of the haem peroxidase-cyclooxygenase superfamily, is abundantly expressed in neutrophils and to a lesser extent in monocytes and certain type of macrophages. MPO participates in innate immune defence mechanism through formation of microbicidal reactive oxidants and diffusible radical species. A unique activity of MPO is its ability to use chloride as a cosubstrate with hydrogen peroxide to generate chlorinating oxidants such as hypochlorous acid, a potent antimicrobial agent. However, evidence has emerged that MPO-derived oxidants contribute to tissue damage and the initiation and propagation of acute and chronic vascular inflammatory disease. The fact that circulating levels of MPO have been shown to predict risks for major adverse cardiac events and that levels of MPO-derived chlorinated compounds are specific biomarkers for disease progression, has attracted considerable interest in the development of therapeutically useful MPO inhibitors. Today, detailed information on the structure of ferric MPO and its complexes with low-and high-spin ligands is available. This, together with a thorough understanding of reaction mechanisms including redox properties of intermediates, enables a rationale attempt in developing specific MPO inhibitors that still maintain MPO activity during host defence and bacterial killing but interfere with pathophysiologically persistent activation of MPO. The various approaches to inhibit enzyme activity of MPO and to ameliorate adverse effects of MPO-derived oxidants will be discussed. Emphasis will be put on mechanism-based inhibitors and high-throughput screening of compounds as well as the discussion of physiologically useful HOCl scavengers.

Excessive hydrogen peroxide is harmful for almost all cell components, so its rapid and efficient removal is of essential importance for aerobically living organisms. Conversely, hydrogen peroxide acts as a second messenger in signal-transduction pathways. H 2 O 2 is degraded by peroxidases and catalases, the latter being able both to reduce H 2 O 2 to water and to oxidize it to molecular oxygen. Nature has evolved three protein families that are able to catalyze this dismutation at reasonable rates. Two of the protein families are heme enzymes: typical catalases and catalase-peroxidases. Typical catalases comprise the most abundant group found in Eubacteria, Archaeabacteria, Protista, Fungi, Plantae, and Animalia, whereas catalase-peroxidases are not found in plants and animals and exhibit both catalatic and peroxidatic activities. The third group is a minor bacterial protein family with a dimanganese active site called manganese catalases. Although catalyzing the same reaction (2 H 2 O 2 → 2 H 2 O + O 2 ), the three groups differ significantly in their overall and active-site architecture and the mechanism of reaction. Here, we present an overview of the distribution, phylogeny, structure, and function of these enzymes. Additionally, we report about their physiologic role, response to oxidative stress, and about diseases related to catalase deficiency in humans.

Myeloperoxidase plays a fundamental role in oxidant production by neutrophils. The enzyme uses hydrogen peroxide to oxidize chloride (Cl-), bromide (Br-), iodide (I-), and the pseudohalide thiocyanate (SCN-) to their respective hypohalous acids. This study for the first time presents transient kinetic measurements of the oxidation of these halides and thiocyanate by the myeloperoxidase intermediate compound I, using the sequential mixing stopped-flow technique. At pH 7 and 15 degrees C, the two-electron reduction of compound I to the native enzyme by Cl- has a second-order rate constant of (2.5 +/- 0.3) x 10(4) M(-1) s(-1), whereas reduction of compound I by SCN- has a second-order rate constant of (9.6 +/- 0.5) x 10(6) M(-1) s(-1). Iodide [(7.2 +/- 0.7) x 10(6) M(-1) s(-1)] is shown to be a better electron donor for compound I than Br- [(1.1 +/- 0.1) x 10(6) M(-1) s(-1)]. The pH dependence studies suggest that compound I reduction by (pseudo-)halides is controlled by a residue with a pKa of about 4.6. The protonation of this group is necessary for optimum (pseudo-)halide anion oxidation. These transient kinetic results are underlined by steady-state spectral and kinetic investigations. SCN- is shown to be most effective in shifting the system myeloperoxidase/hydrogen peroxide from the peroxidatic cycle to the halogenation cycle, whereas iodide is shown to be more effective than bromide which in turn is much more effective than chloride. Decreasing pH increases the rate of this transition. Our results show that thiocyanate is an important substrate of myeloperoxidase in most environments and that hypothiocyanate is likely to contribute to leukocyte antimicrobial activity.

No abstract

Chlorite dismutase (Cld) is a unique heme enzyme catalyzing the conversion of ClO 2؊ to Cl ؊ and O 2 . Cld is usually found in perchlorate-or chlorate-reducing bacteria but was also recently identified in a nitriteoxidizing bacterium of the genus Nitrospira. Here we characterized a novel Cld-like protein from the chemolithoautotrophic nitrite oxidizer Nitrobacter winogradskyi which is significantly smaller than all previously known chlorite dismutases. Its three-dimensional (3D) crystal structure revealed a dimer of two identical subunits, which sharply contrasts with the penta-or hexameric structures of other chlorite dismutases. Despite a truncated N-terminal domain in each subunit, this novel enzyme turned out to be a highly efficient chlorite dismutase (K m ‫؍‬ 90 M; k cat ‫؍‬ 190 s ؊1 ; k cat /K m ‫؍‬ 2.1 ؋ 10 6 M ؊1 s ؊1 ), demonstrating a greater structural and phylogenetic diversity of these enzymes than was previously known. Based on comparative analyses of Cld sequences and 3D structures, signature amino acid residues that can be employed to assess whether uncharacterized Cld-like proteins may have a high chlorite-dismutating activity were identified. Interestingly, proteins that contain all these signatures and are phylogenetically closely related to the novel-type Cld of N. winogradskyi exist in a large number of other microbes, including other nitrite oxidizers.

Graphical abstract

Myeloperoxidase (MPO) is a major neutrophil protein and may be involved in the nitration of tyrosine residues observed in a wide range of inflammatory diseases that involve neutrophils and macrophage activation. In order to clarify if nitrite could be a physiological substrate of myeloperoxidase, we investigated the reactions of the ferric enzyme and its redox intermediates, compound I and compound II, with nitrite under pre-steady state conditions by using sequential mixing stopped-flow analysis in the pH range 4 -8. At ؊1 at pH 5. pH dependence studies suggest that both complex formation between the ferric enzyme and nitrite and nitrite oxidation by compounds I and II are controlled by a residue with a pK a of (4.3 ؎ 0.3). Protonation of this group (which is most likely the distal histidine) is necessary for optimum nitrite binding and oxidation.